CN113621601B

CN113621601B - Sucrose isomerase mutant, coding gene and application thereof

Info

Publication number: CN113621601B
Application number: CN202110706337.4A
Authority: CN
Inventors: 柳志强; 张烽; 蔡雪; 程峰; 郑裕国
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Henan Zhongyuan Yuze Biotechnology Co ltd
Priority date: 2021-06-24
Filing date: 2021-06-24
Publication date: 2023-06-30
Anticipated expiration: 2041-06-24
Also published as: CN113621601A

Abstract

The invention belongs to the technical field of biology, and particularly relates to a sucrose isomerase mutant from Erwiniasp.Ejp617, a coding gene thereof, a recombinant vector containing the mutant gene and application of the sucrose isomerase mutant in preparing isomaltulose by biocatalysis of sucrose. The amino acid sequence of the sucrose isomerase mutant is shown as SEQ ID NO. 1. The invention constructs a sucrose isomerase double mutant Q209S/R456H with improved enzyme activity and catalysis efficiency, and the enzyme activity of the sucrose isomerase mutant Q209S/R456H reaches 684U/mg in water bath with pH of 6.0 and 30 ℃, and the catalysis efficiency is more than 16 times of that of wild sucrose isomerase; enzyme kinetic analysis shows that Q209S/R456H K _m The values were respectively reduced by 48.7% and k compared with the native enzyme _cat The value is 8.3 times of that of the natural enzyme; catalytic efficiency k _cat /K _m Is more than 16 times of the WT. When the sucrose is catalyzed to produce isomaltulose, the maximum conversion rate of the isomaltulose of the mutant is improved by 19.3 percent compared with that of the natural enzyme.

Description

Sucrose isomerase mutant, coding gene and application thereof

Field of the art

The invention belongs to the technical field of biology, and particularly relates to a sucrose isomerase mutant from Erwiniasp.Ejp617, a coding gene thereof, a recombinant vector containing the mutant gene and application of the sucrose isomerase mutant in preparing isomaltulose by biocatalysis of sucrose.

(II) background art

Isomaltulose (isomaltose), also known as Palatinose (Palatinose), is a reducing disaccharide, an isomer of sucrose, composed of D-glucose and D-fructose joined by an alpha-1, 6 glycosidic bond, unlike the alpha-1, 2 glycosidic bond in sucrose. In 1957, it was first discovered by Weienhagen et al in beet production. Isomaltulose has a sweetness profile and mouthfeel similar to sucrose, but its sweetness is low, only 52% of sucrose, and its outstanding advantages compared to sucrose are mainly represented by: (1) low cariogenic properties; (2) Has good therapeutic effect on diabetic and pre-diabetic patients; (3) Selectively stimulating the growth of bifidobacteria in the human intestinal tract; (4) Very low hygroscopicity, strong stability and longer shelf life; (5) Is suitable for the people such as middle and primary school students, white collar people and the like who need to continuously and long-time mental work. Isomaltulose has been widely used as a promising functional sweetener in japan, the united states, western europe, etc., and has a range of applications including hard candy, soft candy, chewing gum, chocolate, baked goods, fruit cans, jams, sports drinks, toothpastes, etc. Isomaltulose is also a starting material for Isomalt (isomart). Isomaltulose alcohol is a functional sugar alcohol which is internationally emerging in recent years and is widely applied to the production of products such as sugarless foods, sugarless health-care products, sugarless medicines and the like.

Isomaltulose is produced by sucrose isomerase EC 5.4.99.22 (Sucrose isomerase), or isomaltulose synthase (Isomaltulose syntheses), sucrose glucosyltransferase (Sucrose glucosylmutase), α -glucosyltransferase (α -glucosyltransferase), which rearranges the α -1,2 linkage of glucose and fructose in sucrose, to produce trehalose when rearranged to α -1,4 linkages, and to isomaltulose when rearranged to α -1,6 linkages. Currently, sucrose isomerase used for isomaltulose production is derived from various microorganisms such as E.coli NX-5, E.coli D12 (Erwinia sp.D12), serratia ATCC15928 (Serratia plymuthica ATCC 15928), klebsiella, pseudomonas acidophilus MX-45 (Pseudomonas mesoacidophila MX-45) and E.coli NCPPB1578 (E.rhapontici NCPPB 1578), natural enzymes from E.coli DSM4484 (GenBank accession number AAK 28735.1), E.coli NX-5 (ADJ 56407.2), E.coli FMB-1 (Enterobacter sp.FMB-1) (ACF 42098.1), pseudomonas acidophilus MX-45 (ACO 05018.1), E.rubrum CBS574.77 (CAF 32985.1), klebsiella pneumoniae NK33-98-8 (Klebsiella pneumonia NK-98-8) (AAM 96902.1), UQ68 (49) and Klebsiella X33-98-8) (AAM 96902.1), klebsiella de (AAK 82938.1) and recombinant enzymes from E.coli LXYZ.sp.5 (ADJ 56407.2). Although sucrose isomerase produced by the above-mentioned bacteria can convert sucrose into isomaltulose, the yield is very unstable and the conversion rate is not high, which is 8% -85%. In addition, besides the main product, partial trehalose and a small amount of byproducts such as isomaltose, isomaltotriose, glucose, fructose and the like exist in the enzyme conversion solution, so that the product specificity is not high. In order to increase the enzyme activity of sucrose isomerase to increase the yield of isomaltulose, the production of isomaltulose high-producing strain by various mutation methods is also a big hot spot of current research. According to Zhang Hongda, klebsiella LX3 screened in a laboratory is taken as an initial strain, the strain is subjected to mutagenesis by using a normal pressure room temperature plasma injection technology, and the saccharose isomerase activity, the isomaltulose content and the detection of the bacterial flocculation effect in fermentation liquor are measured to obtain a strain (LX 3-1) with high isomaltulose yield and lower viscosity, compared with a wild strain, the enzyme activity of the saccharose isomerase of a mutant strain is improved by 20.42% (P < 0.05), the yield of the isomaltulose is improved by 41.87%, and after 6 subcultures of the mutant strain, the enzyme activity of the saccharose isomerase and the yield of the isomaltulose in the fermentation liquor are still stable. Liu et al mutated Y296 and Q299 of sucrose isomerase derived from Pantoea dispersible UQ68J near the substrate binding site, to achieve improved isomaltulose production.

At present, although there are many researches on sucrose isomerase at home and abroad, most of the researches remain at laboratory level, and further researches on the industrial application of sucrose isomerase are needed.

(III) summary of the invention

The invention aims to provide a sucrose isomerase mutant, a coding gene thereof, a recombinant vector containing the mutant gene and application of the sucrose isomerase mutant in preparing isomaltulose by biologically catalyzing sucrose.

The technical scheme adopted by the invention is as follows:

a sucrose isomerase mutant has an amino acid sequence shown in SEQ ID NO. 1. The sucrose isomerase mutant is obtained by mutating the 209 th amino acid from Erwiniasp. Ejp617 from glutamine to serine and mutating the 456 th arginine to histidine. The invention uses error-prone PCR technology to carry out site-directed mutagenesis on sucrose isomerase for molecular transformation, thereby further improving the enzyme activity of sucrose isomerase and the yield of isomaltulose.

The invention also relates to a gene encoding the sucrose isomerase mutant.

Specifically, the nucleotide sequence of the coding gene is shown as SEQ ID No. 2.

The invention also relates to a recombinant vector containing the coding gene. These genes, expression cassettes, plasmids, transformants can be obtained by genetic engineering construction methods well known to those skilled in the art.

The invention also relates to application of the sucrose isomerase mutant in preparing isomaltulose by biocatalysis of sucrose.

When used as biocatalysts for production, the sucrose isomerase mutants of the present invention may take the form of enzymes or bacteria. The enzyme forms include free enzymes, immobilized enzymes, including purified enzymes, crude enzymes, fermentation broths, vector immobilized enzymes, cell debris, etc.: the forms of the bacterial cells include viable bacterial cells and dead bacterial cells.

Specifically, the application is as follows: the sucrose isomerase mutant is used as a catalyst, sucrose is used as a substrate, the reaction is carried out for 3 to 12 hours at the pH of 6.0 to 7.0 and the temperature of 28 to 32 ℃, and the isomaltulose is obtained in the reaction liquid.

Preferably, the concentration of sucrose in the reaction system is 500-600 g/L.

Compared with the prior art, the invention has the beneficial effects that: the invention constructs a sucrose isomerase double mutant Q209S/R456H with improved enzyme activity and catalysis efficiency, and the enzyme activity of the sucrose isomerase mutant Q209S/R456H reaches 684U/mg in water bath with pH of 6.0 and 40 ℃, and the catalysis efficiency is more than 16 times of that of wild sucrose isomerase; enzyme kinetic analysis shows that Q209S/R456H K _m The values respectively decrease compared with the natural enzymes48.7%, k _cat The value is 8.3 times of that of the natural enzyme; catalytic efficiency k _cat /K _m Is more than 16 times of the WT. When the sucrose is catalyzed to produce isomaltulose, the maximum conversion rate of the isomaltulose of the mutant is improved by 19.3 percent compared with that of the natural enzyme.

(IV) description of the drawings

Fig. 1: the method is to obtain good mutation point color reaction by using a high-throughput screening method;

fig. 2: is natural sucrose isomerase and five mutant pure enzyme SDS-PAGE gel electrophoresis; wherein M represents a protein molecular weight standard, WT is wild-type sucrose isomerase, and lane 1 is mutant Q209N; lane 2 is mutant R456K; lane 3 is mutant Q209S, lane 4 is mutant R456H, and lane 5 is mutant Q209S-R456H.

Fig. 3: optimum temperature and pH values for ErSIase_WT and ErSIase_Q209S-R456H. (A) optimum temperature of the ErSIase_WT, (B) optimum pH of the ErSIase_WT, (C) optimum temperature of the ErSIase_Q209S-R456H, and (D) optimum pH of the ErSIase_Q209S-R456H.

Fig. 4: the conversion of isomaltulose was prepared for the wild-type sucrose isomerase and its mutants at 40 ℃.

Fig. 5: construction of recombinant E.coli BL21 (DE 3)/pET 28b (+) -ErSIase containing recombinant plasmid of ErSIase gene.

(fifth) detailed description of the invention

The present invention will be described in further detail with reference to the following specific examples, but the present invention is not limited to the following examples:

the invention relates to the addition amount, content and concentration of various substances, wherein the percentage content refers to the mass percentage content unless specified otherwise.

Reagents for upstream genetic engineering operations: the one-step cloning kits used in the examples of the present invention were all purchased from Vazyme, nanjinouzan biotechnology Co., ltd; plasmid extraction kit DNA recovery purification kits were purchased from Axygen hangzhou limited; plasmids and the like are purchased from Shanghai; DNA marker, fast Pfu DNA polymerase, low molecular weight standard protein, agarose electrophoresis reagent, primer synthesis and gene sequencing and gene synthesis work are completed by catalpa and Xeong biotechnology limited company of Hangzhou qing. The above methods of reagent use are referred to in the commercial specifications. Common reagents such as sucrose and isomaltulose are purchased from national pharmaceutical group chemical company, ltd.

Example 1: cloning and expression of the sucrose isomerase gene in E.coli BL21 (DE 3).

Constructing an expression vector: the sucrose isomerase gene synthesis described below was performed by catalpa, biotechnology limited, of qinghao. The sucrose isomerase gene (GenBank: G37835) derived from Erwinia (Erwiniasp. Ejp 617) was seamlessly cloned between the Nco I and Xho I of the pET28b (+) vector by PCR to obtain the expression vector pET28b (+) -SI with sucrose isomerase. The PCR procedure was as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95℃for 15s, annealing at 53-58℃for 15s, extension at 72℃for 1.5 min for 25 cycles; then extended at 72℃for 10 minutes.

The preparation method of the competent cells comprises the following steps: e.coli BL21 (DE 3) strain preserved in glycerol tubes is obtained from a refrigerator at the temperature of minus 80 ℃, streaks are formed on an antibiotic-free LB plate, and the strain is cultured for 10 hours at the temperature of 37 ℃ to obtain single colony; picking single colony of LB plate, inoculating into test tube containing 5mL LB culture medium, culturing at 37deg.C and 180rpm for 9h; 200 mu L of bacterial liquid is taken from a test tube and inoculated into 50mL of LB culture medium, OD is cultivated at 37 ℃ and 180rpm ₆₀₀ To 0.4-0.6; precooling the bacterial liquid on ice, taking the bacterial liquid into a sterilized centrifuge tube, placing the bacterial liquid on the ice for 10min, and centrifuging the bacterial liquid at 4 ℃ for 10min at 5000 rpm; pouring out supernatant, taking care to prevent contamination, pre-cooling with 0.1mol/L CaCl ₂ The precipitated cells were resuspended in aqueous solution and placed on ice for 30min; centrifuging at 4deg.C and 5000rpm for 10min, discarding supernatant, and pre-cooling with 0.1mol/L CaCl containing 15% glycerol ₂ The precipitated cells were resuspended in aqueous solution, 100. Mu.L of the resuspended cells were dispensed into sterilized 1.5mL centrifuge tubes and stored in a-80℃freezer and removed as needed.

Constructing an engineering bacteria library: e.coli BL21 (DE 3) (Invitrogen) competent cells stored at-80℃were ice-bathed at 0℃for 10min, then 5. Mu.L of pET28b (+) -SI with sucrose isomerase gene expression vector was added in a super clean bench, respectively, ice-bathed at 0℃for 30min, hot-shocked at 42℃in water bath, ice-bathed at 0℃for 4min, 600. Mu.L of LB medium was added, and shaking culture was performed at 37℃and 200rpm for 1h; coating on LB plate containing 50 mug/ml kanamycin resistance, culturing at 37 ℃ for 8-12h to obtain recombinant E.coli BL21 (DE 3)/pET 28b (+) -SI engineering bacteria containing expression recombinant plasmid.

Example 2: induction expression and purification of sucrose isomerase recombinant bacteria

Wet cell containing sucrose isomerase: the recombinant E.coli BL21 (DE 3)/pET 28b (+) -SI obtained in example 2 was inoculated into LB liquid medium containing 50. Mu.g/mL kanamycin resistance, cultured at 37℃and 200rpm for 12 hours, inoculated into fresh LB liquid medium containing 50. Mu.g/mL kanamycin resistance in an inoculum size of 1% (v/v), and cultured at 150rpm to cell OD at 37℃ ₆₀₀ Adding IPTG with the final concentration of 0.1mM to 0.6-0.8, performing induction culture at 25 ℃ for 10 hours, centrifuging at 4 ℃ and 8000rpm for 20 minutes, discarding supernatant, collecting precipitate, and washing twice with Tris HCl buffer solution with the pH of 7.0 and 50mM to obtain a recombinant strain E.coli BL21 (DE 3)/pET 28b (+) -SI wet bacterial strain containing sucrose isomerase; the wet cells were resuspended in 50mM Tris HCl buffer pH 7.0 and sonicated on an ice-water mixture for 20min under sonication conditions: the power was 400W, the mixture was broken for 1s and suspended for 5s to obtain a crude enzyme solution.

Purification of sucrose isomerase: since 6 histidine tags are expressed together with the sucrose isomerase gene, the peptide fragment and divalent Ni that can be expressed by this histidine tag ²⁺ Is purified by affinity chromatography using Ni affinity columns (40X 12.6mm, bio-Rad, USA). The purification process is mainly used and performed. The specific operation is as follows: (1) buffer A (50 mM NaH) was equilibrated with 5 column volumes of Ni column ₂ PO ₄ ·2H ₂ The Ni column was equilibrated with O+300mM NaCl+50mM imidazole to baseline stability; (2) sample loading, wherein the flow rate is 1mL/min, the loading amount is 25-40mg/mL of protein, and the target protein is adsorbed on a Ni column; (3) flushing the hybrid protein with buffer A with 6 times of column volume, and stabilizing the flow rate to a baseline at 1 mL/min; (4) with eluent B (50 mM NaH ₂ PO ₄ ·2H ₂ O+300mM NaCl+500mM imidazole) at a flow rate of 1mL/min, and collecting the target protein. The target protein was dialyzed overnight in a phosphate buffer solution (pH 7.5, 20 mM), and purified enzyme was obtained, and the SDS-PAGE pattern was shown in FIG. 2.

Example 3 construction and screening of sucrose isomerase Gene library.

(one) introduction of beneficial mutations by error-prone PCR

Plasmid DNA was isolated from E.coli BL21 (DE 3)/pET 28b (+) -SI recombinant E.coli using the MiniBEST plasmid purification kit (TaKaRa, dalian, china). In the presence of 60ng of plasmid (pET 28 b-SI) as template, 60pmol of each mutagenesis primer (Table S1), 0.4mM MnCl ₂ Amplification of DNA fragments Taq PCR Starmix and Loding Dye (TaKaRa, daidan, china) was performed in 50. Mu.L of the reaction mixture of 2X. The amplification process of the gene encoding the SIase is: 94℃for 2min, followed by 30 cycles of 94℃30s,60℃30s and 72℃2min, respectively, and finally 10min at 72 ℃. This protocol resulted in an average mutation frequency of every 1000bp

And (3) substitution of a single base. The PCR product was digested with QuickCut Dpn I at 37℃for 3 hours to remove the template. The library gene fragments were then purified using MiniBEST DNA fragment purification kit (TaKaRa, dalian, china). The purified gene fragment was then ligated into pET28b between the Nco I and Xho I sites to form recombinant plasmid pET28b-SI. pET28b-SI was then transformed into E.coli and all transformed cells were grown overnight. The best variants were screened and sequenced.

Table S1: error-prone PCR primer sequences

(II) establishment of high throughput screening method and detailed procedure

1. Preparing seed liquid by 96 deep hole plate culture: 500. Mu.L of LB liquid medium containing 0.1% (v/v) 100g/LKana was added to each well of the 96-deep well plate, then single colonies were picked up with sterile toothpicks into the corresponding wells, and the remaining 4 wells were selected from each 96-deep well plate, and WT single colonies were picked up and added as positive controls. Then, the mixture was sealed with a cap, and cultured at 37℃and 180rpm with shaking for 5 hours.

2. Seed liquid transfer and induction: taking 50 mu L of the seed liquid of the transformant cultured in the previous step into another 96 deep well plate, adding 900 mu L of LB liquid medium containing 0.1% (v/v) 100g/L Kan, sealing by a cover, culturing for 2h at 37 ℃ with shaking at 180rpm, adding 50 mu L of LB liquid medium containing 0.8% (v/v) 120g/L IPTG and 0.1% (v/v) 100g/L Kana into each well, sealing by the cover, and culturing for more than 10h with shaking at 25 ℃ and 180 rpm. The remaining seed solution of the transformant was kept in a refrigerator at 4℃for further use.

3. Enzymatic activity detection by DNS method: mu.L of the induced solution was taken in a 96-well plate, and 50. Mu.L of 50g/L sucrose (prepared with 50mM citric acid-disodium hydrogen phosphate buffer (pH 6.0)) was added thereto, and reacted at a constant temperature of 40℃for 15 minutes. Then 50 mu L of DNS reagent is added, the color is developed by heating for about 25s with medium fire in a microwave oven, the experimental result is observed, and the absorbance of the developing solution at 540nm is detected by an enzyme-labeled instrument.

4. Preserving and sequencing high-enzyme activity mutant bacterial liquid: according to the determination result of the DNS method, labeling a sample with higher absorbance than that of the positive control, taking 200 mu L of the corresponding seed liquid in a glycerol tube, adding 200 mu L of 50% glycerol, uniformly mixing, and then storing in a refrigerator at the temperature of minus 20 ℃. Then 10 mu L of seed solution is taken and cultured in 10mL of LB liquid medium containing 0.1% (v/v) 100g/L Kan at 37 ℃ under shaking at 180rpm for more than 7 hours, more than 200 mu L of bacterial solution is taken and tested, and proper mutants are reserved according to the sequencing result.

And (III) carrying out re-screening by using a High Performance Liquid Chromatography (HPLC) method

High Performance Liquid Chromatography (HPLC) to determine enzyme activity: 200. Mu.L of the crude enzyme solution (100 g/L of the supernatant after ultrasonic disruption of wet cells) was added to 800. Mu.L of 50mM citric acid-disodium hydrogen phosphate buffer (pH 6.0) containing sucrose (sucrose final concentration: 50 g/L). After 30min of reaction at 40 ℃, the mixture is treated by boiling water bath for 10min for inactivation. The centrifuged supernatant was diluted 5-fold with a mobile phase, filtered through a filter membrane, and the isomaltulose content in the diluted solution was measured by high performance liquid chromatography. Liquid chromatography usedThe instrument is waters2414; the mobile phase is acetonitrile and water mixed solution with the volume ratio of 4:1, and the flow rate is 1.5mL/min; the chromatographic column is special for Agilent ZORBAX sugar analysis (specification 4.6X1250 mm, carbon loading 3.5%, pore diameter 70 angstrom, particle diameter 5 μm, specific surface area 300 m) ² /g, pH range 2.0-8.0); the detector is a differential refraction detector; the external temperature and the column temperature are 35 ℃ and 30 ℃ respectively; the sample loading was 10. Mu.L. Calibration of a high performance liquid chromatography standard curve: sucrose solutions and isomaltulose solutions of different concentrations (1 mM, 10mM, 20mM, 40mM, 80mM and 120 mM) were prepared, the peak areas thereof were measured by HPLC methods, respectively, and the sucrose solutions (or isomaltulose solutions) were plotted on the abscissa and the peak areas on the ordinate, respectively, to draw the relevant standard curves.

Definition of enzyme Activity: the amount of enzyme required to catalyze sucrose isomerization to 1. Mu. Mol isomaltulose in 1min at pH6.0 and 40℃is 1 enzyme activity unit (U).

The calculation formula of the specific activity of the pure enzyme is as follows:

after 4500 mutants were screened, two optimal mutants Q209N, R456K, E.coli BL21 (DE 3)/pET 28b (+) -SI-Q209N and E.coli BL21 (DE 3)/pET 28b (+) -SI R456K were obtained with the enzyme activities reaching 59U/mg,165U/mg, respectively, whereas the wild type E.coli BL21 (DE 3)/pET 28b (+) -SI enzyme activity was 39U/mg protein, the specific activities of mutants Q209N and R456K were increased by 1.5 and 4.2 times, respectively.

Example 4: site-directed saturation mutation of sucrose isomerase

In order to further screen potential activity-improving strains, the two beneficial mutation sites Q209 and R456 of the sucrose isomerase obtained in example 3 are subjected to site-directed saturation mutation, and further screening is carried out, wherein PCR primer design is shown in Table 1, and a PCR system (50 mu L) is as follows: 2.times.Phanta Max buffer 25. Mu.L, dNTPs 1. Mu.L, mutation upper and lower primers 1. Mu.L each, template (starting strain) 1. Mu.L, pfu DNA polymerase 0.5. Mu.L, and ddH ₂ O to 50. Mu.L. The PCR conditions were: pre-denaturation at 95 ℃ for 3min: denaturation at 95℃for 15s, annealing at 60℃for 15s, extension at 72 ℃Stretching for 7min for 20s, and circulating for 30 times; final extension at 72℃for 10min. The PCR product is verified by DNA agarose gel electrophoresis, and after passing through DpnI digestion template, the PCR product is transformed into E.coli BL21 (DE 3) competent cells, the transformed product is coated on LB plate containing 50 mug/mL ampicillin resistance, the inversion culture is carried out at 37 ℃ for overnight, the obtained mutant is screened for dominant mutant, and the obtained dominant strain is sent to Hangzhou department of the biological technology of the field of China for sequencing confirmation and preservation.

Table 1: sucrose isomerase site-directed saturation mutation primer design

Liquid phase detection the enzyme activity was calculated as in example 3 above.

After site-directed saturation mutation of the two beneficial mutation sites Q209 and R456 of sucrose isomerase, it was found that the enzyme activities of mutants Q209S and R456H were increased 7.3-fold and 11.5-fold, respectively, with the exception of Q209N and R456K as beneficial mutation sites, with a greater magnitude than that of mutants Q209N and R456K. Then, the Q209N and R456K mutants are combined to obtain a double mutant E.coli BL21 (DE 3)/pET 28b (+) -SI-Q209S/R456H, the enzyme activity reaches the maximum of 684U/mg, and the catalytic efficiency reaches the maximum of 22.67S ^-1 mM ^-1 Is more than 16 times of WT.

Table 2: enzymatic characterization of wild-type and different mutant sucrose isomerase

Example 5: the substrate sucrose is catalyzed to synthesize isomaltulose by using a recombinant E.coli BL21 (DE 3)/pET 28b (+) -SI-Q209S/R456H sucrose isomerase mutant.

The temperature and pH values of the original thalli and the mutant E.coli BL21 (DE 3)/pET 28b (+) -SI-Q209S/R456H are optimized. The reaction was performed at different pH (from 4.6 to 10.0) to determine its optimal pH, and the buffers used at different pH conditions were: 100mM disodium hydrogen phosphate buffer (pH 4.6-7.5), 100mM Tris-HCl buffer (pH 7.5-8.6), and 100mM Gly medium-NaOH buffer (pH 8.6-10.0). The optimum temperature of the ErSIase was also studied by measuring the enzyme activity at different temperatures (20-55 ℃).

The results showed that the optimal pH values for ErSIase_WT and ErSIase_Q209S-R456H were 6.5 and 6.0, respectively (FIGS. 3B and 3D). As shown in FIGS. 3A and 3C, both purified ErSIase_WT and ErSIase_Q209S-R456H had the highest enzymatic activity at around 40 ℃.

And finally, carrying out catalytic reaction by using whole cells under the optimal temperature and pH reaction conditions. The reaction system is 600g/L sucrose, recombinant E.coli BL21 (DE 3)/pET 28b (+) -SI-Q209S/R456H (high expression level) (0.1 g/L stem cell). Reaction conditions: constant temperature water bath 40 ℃, rotating speed 600rpm. During the whole reaction, samples (100. Mu.L) were taken at intervals of 30min, 5. Mu.L of 6M concentrated hydrochloric acid was added to terminate the reaction, and then the samples were treated, and the conversion was determined by measuring the sucrose or isomaltulose concentration using HPLC, and the reaction progress curve is shown in FIG. 4.

When the concentration of the substrate sucrose is 600g/L, the conversion rate reaches 80.0% at 60min by using 0.1g/L catalyst, and the conversion rate reaches 93.6% after the reaction is completed in 3 hours finally. Under the same reaction conditions, the conversion rate was only 74.3% after completion of the reaction for 3 hours using the initial cell (sucrose isomerase WT). Compared with wild-type sucrose isomerase, the conversion rate of the E.coli BL21 (DE 3)/pET 28b (+) -SI-Q209S/R456H sucrose isomerase mutant is improved by 19.3 percent when the mutant catalyzes 600g/L sucrose substrate.

Sequence listing

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<400> 2

atgtctcgtt tcaccctgtc taccgttatg gctctgctgg ttgctccggt tctgtctgtt 60

ctgccgggtc aggttatggc tggtaaaatg gacatggcta ccacccagct ggctcagaaa 120

tctgacgact tcccggcgtg gtggaaacag gcggtcttct accaggtgta cccgcgctct 180

ttcaaagaca ccaacggtga cggtatcggt gacctgaaag gtatcatcga aaaactggac 240

tacctgaaac gtctgggtgt tgacgctatc tggatcaacc cgcactacga ctctccgaac 300

accgacaacg gttacgacat ccgtgactac cgtaaaatca tgaaagaata cggtaccatg 360

gaagacttcg accgtctgat cgctgaaatg aacaaacgta acatgcgtct gatgatcgac 420

atcgttatca accacacctc tgaccagcac tcttggttcg ttcagtctaa aggttctaaa 480

gacaacccgt accgtgacta ctacttctgg cgtgacggta aaaacggtca gccgccgaac 540

aactacccgt ctttcttcgg tggttctgct tggaaaaaag aagacaactc tggtcagtac 600

tacctgcact acttcgctac ccagagtccg gacctgaact gggacaaccc gaaagttcgt 660

gaagacctgt acgctatgct gcgtttctgg ctggacaaag gtgttgctgg tctgcgtttc 720

gacaccgttg ctacctacgc taaaatcccg ggtttcccgg acctgacccc gcagcagcgt 780

aaaaacttcg ctcgtaccta caccgaaggt ccgtctatcc accgttacat caaagaaatg 840

caccagcagg ttttctctca ctacaacatc gctaccgctg gtgaaatctt cggtgttccg 900

ctggaaaaat ctatcgacta cttcgaccgt cgtcgtggtg aactgaacat cgctttcacc 960

ttcgacctga tccgtctgga ccgtggtgtt gaagaacgtt ggcgtcagaa agcttggtct 1020

ctgaccgact tccgtcagac catcgacaaa gttgaccgtg ttgctggtaa atacggttgg 1080

aacgctttct tcctggacaa ccacgacaac ccgcgtgctg tttctcactt cggtgacgac 1140

cgtccgcagt ggcgtcaggc ttctgctaaa gctctggcta ccctgatgat cacccagcgt 1200

gctaccccgt tcatctacca gggttctgaa ctgggtatga ccaactaccc gttcaaatct 1260

atcgctgact tcgacgacat cgaggttaaa ggcttctggc aggactacgt ttcttctggt 1320

aaagttgacc cggaagaatt catgcgtaac gttcgtctga cctctcatga caactctcgt 1380

accccgttcc agtgggacga atctgctaac gctggtttca cctctggtac cccgtggttc 1440

aacgttaacc cgaactacaa actgatcaac gctgctgacc agacccgtga cccggactct 1500

gttttcaact actaccgtaa actgatcggt ctgcgtcacg ctatcccggc tctgacctac 1560

ggtgaataca aagacctgga cccgaacaac gacaccgttt acgctttcac ccgtacccac 1620

ggtgacaaac gttacctggt tgttatcaac ttcaaagaaa acgttgttaa ctaccgtctg 1680

ccggaccagc tgaccatccg tcagaccctg tctgaatctt ctgctatcca gccggttgct 1740

gaaaacgctc gtgaactgct gctgcagccg tggcagtctg gtatctacca gctgaac 1797

<210> 3

<211> 39

<212> DNA

<213> Unknown (Unknown)

<400> 3

gaaggagata taccatgtct cgtttcaccc tgtctaccg 39

<210> 4

<211> 35

<212> DNA

<213> Unknown (Unknown)

<400> 4

tggtgctcga gttggttcag ctggtagata ccaga 35

<210> 5

<211> 33

<212> DNA

<213> Unknown (Unknown)

<400> 5

tacttcgcta cccagnnkcc ggacctgaac tgg 33

<210> 6

<211> 33

<212> DNA

<213> Unknown (Unknown)

<400> 6

ccagttcagg tccggmnnct gggtagcgaa gta 33

<210> 7

<211> 33

<212> DNA

<213> Unknown (Unknown)

<400> 7

gttcgtctga cctctnnkga caactctcgt acc 33

<210> 8

<211> 33

<212> DNA

<213> Unknown (Unknown)

<400> 8

ggtacgagag ttgtcmnnag aggtcagacg aac 33

Claims

1. A sucrose isomerase mutant has an amino acid sequence shown in SEQ ID NO. 1.

2. A gene encoding the sucrose isomerase mutant of claim 1.

3. The coding gene according to claim 2, wherein the nucleotide sequence of the coding gene is shown in SEQ ID No. 2.

4. A recombinant vector comprising the coding gene of claim 2.

5. Use of the sucrose isomerase mutant according to claim 1 for biocatalytic sucrose preparation of isomaltulose.

6. The application of claim 5, wherein the application is: the sucrose isomerase mutant is used as a catalyst, sucrose is used as a substrate, the reaction is carried out for 3 to 12 hours under the conditions of pH of 6.0 to 7.0 and 28 to 32 ℃, and the isomaltulose is obtained in the reaction liquid.

7. The method according to claim 5, wherein the sucrose concentration in the reaction system is 500 to 600g/L.